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New Developments in Multiplexing Aim To Reduce Immunoassay Variability

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Immunoassays are used for a wide variety of proteomic and genomic applications in drug discovery and development, including monitoring toxicity, detecting drug response biomarkers, evaluating therapeutic responses in a clinical trial, and more.

Despite their prevalence, even the best practitioners struggle to run every single immunoassay exactly the same way. Run-to-run variability caused by minute differences among operators, product variation in buffers and reagents, or even small changes in a lab’s environmental conditions has been an ongoing challenge for drug discovery and development teams.

Balancing throughput, specificity and reproducibility in immunoassays 

Part of the problem is due to the serial nature of immunoassays. Whenever results are generated from separate reactions or separate wells, there is a possibility of introducing confounding variables. For many applications, especially high-throughput ones, multiplexing has offered consistency and has allowed scientists to significantly improve the reproducibility of their assays. However, there are still many applications where analytes must be interrogated separately – especially when issues like cross-reactivity must be factored into the equation.

The COVID-19 pandemic has brought this challenge front and center for many researchers. Measuring isotypes of antibodies against the SARS-CoV-2 virus – a process that should be straightforward and reproducible – has been hindered by technical protocols that require the detection of these isotypes by a single reporter in separate reactions. Results from these methods are not always as comparable as scientists would like.

In an ideal scenario, antibody isotypes and other closely related analytes would be measured in the same reaction or well to avoid any possible variability inherent in separate reactions. Using a single-reaction approach would also reduce sample volume requirements and experimental costs since all data points would be produced from a single reaction.

The advantages of multiplexing over single-plex methods

For years now, scientists have adopted multiplex assays to generate more robust and reproducible results compared to separate, single-plex immunoassays like
ELISAs. There are a few different approaches to multiplexing, but in general, multiplex assays improve the quality of your results by testing many targets or analytes in a single reaction.1,2 Additionally, using identical experimental conditions for all of the analytes you test makes the results generated between analytes more easily comparable.

Depending on the multiplex approach used, other advantages of these higher-throughput systems often include miniaturized reactions that lower sample input requirements and reduce reagent use. They also minimize hands-on time, allowing scientists to move on to other tasks, and reduce the cost of each experiment. Importantly, they may detect hundreds of analytes in a single run.

Recently, a new feature of multiplexing technology has been developed to reduce variability in immunoassays further. The dual reporter feature, which uses two reporter channels, allows scientists to collect two different measurements from each analyte in a sample. This would be particularly helpful when the measurements come from related elements, such as detecting antibody isotypes, the presence or absence of post-translational modifications in nucleic acids or proteins, or free versus bound drug in a sample.

In a recent study, a multiplex approach was used to detect both IgG and IgM antibodies to three antigens of the SARS-CoV-2 virus (the spike protein, the receptor-binding domain and the nucleocapsid protein).
3 The technique involved a bead-based multiplexing system in which the dual reporter channels captured two distinct signals from each individual bead, effectively doubling the data produced from the study and ensuring that experimental conditions for both antibody isotypes were identical. Data generated with this method provided a more accurate and complete view of the immune response to the virus compared to traditional methods.

Factors to consider when developing a multiplex assay

With several options for multiplexing immunoassays, there are many key factors to consider when selecting the best option for your lab or experiment:


  • Size: With bench space at a premium, the overall footprint of the system can be important. For the most compact options, look for systems with integrated touchscreens – these save bench space by eliminating the need for separate monitors, keyboards and other peripherals.

  • Capacity: Some labs may need to interrogate a dozen analytes while others need to multiplex hundreds of analytes for each sample. Be sure to assess not only your current multiplexing needs, but also how you anticipate your needs changing in the future to ensure that the platform you choose will continue to support your research for years.

  • Reaction type: Commercially available multiplexing platforms may run reactions in solid state – often binding materials to a slide or plate – or in suspension. Certain sample types might perform better in one type or another, so it’s an important element to check.

  • Dual reporter capability: If the ability to directly compare two different signals for any given analyte would be useful in your research, consider looking for this feature. Having two reporter channels is helpful for immune response studies (antibody isotyping), pharmacology studies (free versus bound drug), and functional studies (comparing methylated to unmethylated nucleic acids, or phosphorylated proteins to non-phosphorylated proteins, for example).


The reproducibility trend

Years ago, scientists in pharmaceutical and biotech companies acknowledged what has come to be known as “
the reproducibility crisis”, which began with the inability to recreate experimental results from in-house studies or from published papers even when carefully following the methods that were originally used.4,5 As the entire biomedical community strives to improve reproducibility, multiplexing has been an important tool for generating more comparable and more carefully controlled immunoassay results.6 Improvements to these multiplex platforms, such as the introduction of two reporter channels, should continue to serve the broader community by delivering more data with less variability.


1. Ray CA, Bowsher RR, Smith WC, et al. Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum. J Pharm Biomed Anal. 2005;36(5):1037–1044. doi: 10.1016/j.jpba.2004.05.024

2. Tighe PJ, Ryder RR, Todd I, Fairclough LC. ELISA in the multiplex era: potentials and pitfalls. Proteomics Clin Appl. 2015;9(3-4):406–422. doi: 10.1002/prca.201400130

3. Angeloni S, Cameron A, Pecora ND, Dunbar, S. A rapid, multiplex dual reporter IgG and IgM SARS-CoV-2 neutralization assay for a multiplexed bead-based flow analysis system. J Vis Exp. 2021;170:e62487. doi: 10.3791/62487

4. Begley C and Ellis L. Raise standards for preclinical cancer research. Nature. 2012;483,531–533. doi: 10.1038/483531a

5. Prinz F, Schlange T, Asadullah K. Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov. 2011;10:712. doi: 10.1038/nrd3439-c1

6. Dias D, Van Doren J, Schlottmann S, et al. Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18. Clin Diagn Lab Immunol. 2005;12(8):959–969. doi: 10.1128/CDLI.12.8.959-969.2005